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a Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan;
b Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan;
c Center for Stem Cell Research and
d Graduate Institute of Cell and Molecular Biology, Taipei Medical University, Taipei, Taiwan
Key Words. Mesenchymal stem cells • Differentiation • Leptin
Correspondence: Shiaw-Min Hwang, Ph.D., Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan. Telephone: 886-3-522-3191; Fax: 886-3-521-4016; e-mail: hsm{at}firdi.org.tw
Received on November 8, 2004;
accepted for publication on September 12, 2005.
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ABSTRACT |
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2 mRNA expression to the same level during adipogenesis in both types of MSCs. These results demonstrate the disparate capacities of MSCs from bone marrow and cord blood and suggest that they be used differently in experimental and therapeutic studies. In addition, the disparate differentiation tendencies of MSCs from different sources should be considered in further applications.
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INTRODUCTION |
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Leptin, a 16-kDa peptide hormone discovered in 1994, is an adipocyte-derived signaling molecule that regulates food intake and increases energy expenditure using specific receptors in the hypothalamus [5, 6]. In adults, leptin is secreted mainly by white adipose tissue into the circulation [5]. Moreover, leptin has also been found to be synthesized and secreted from placental trophoblasts into the maternal and fetal circulation in pregnant women [7]. Current evidence suggests that leptin can affect the function of mesenchyme-lineage cells, such as muscle cells [8], chondrocytes [9], osteoblasts [10], and adipocytes [11]. However, it is still unknown if MSCs are the targets of leptin.
Although the immunophenotypic profiles of MSCs from different sources are very similar [2, 3], knowing if these MSCs express quantitative differences in differentiation potentials is important. To investigate the difference in the functional specificity of osteogenesis and adipogenesis between different sources of MSCs, human bone marrow–derived MSCs (bmMSCs) and umbilical cord blood–derived MSCs (cbMSCs) were compared. Differences in differentiation potentials in osteogenesis and adipogenesis were detected under the same induction conditions. Interestingly, cbMSCs exhibit a significantly stronger osteogenic potential but lower adipogenic capacity than bmMSCs. Leptin was found to directly affect the mesenchyme-lineage differentiation potentials of both types of MSCs tested. Data from our study suggest that the diverse functional potentials of MSCs from different sources should play an important role in their further applications.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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-MEM; Hyclone; Logan, UT; http://www.hyclone.com), containing 20% fetal bovine serum (FBS; Hyclone), 4 ng/ml basic fibroblast growth factor (R&D Systems; Minneapolis; http://www.rndsystems.com), 100 units/ml penicillin, and 100 µg/ml streptomycin (Sigma), and cultured at 37°C in a humidified atmosphere with 5% CO2. The nonadherent cells were removed using a medium change after 3 days of seeding, and the medium was changed twice a week thereafter for 2–3 weeks. When well-developed colonies of fibroblast-like cells appeared, cells were washed with PBS, harvested with 0.05% trypsin-EDTA (Sigma) and passed into new T75 flasks. This protocol was approved by the Institutional Review Board of Taipei Medical University, Taiwan.
Immunophenotyping of MSCs
Cells at passage four were trypsinized, washed, and resuspended in PBS at a concentration of about 100,000 cells/ml. After fixing with 1% methanol at 4°C for 10 minutes, cells were blocked in 1% bovine serum albumin (BSA; Sigma) plus 0.1% FBS for 1 hour at room temperature, then washed with three volumes of PBS. The cell pellet was resuspended in 0.5 ml of primary antibody solution containing 1% BSA plus 0.1% FBS for 40 minutes at 4°C, then washed three times in PBS. Cells were immunolabeled with the following mouse anti-human antibodies: CD34, CD45 (Miltenyi Biotech; Auburn, CA; http://www.miltenyibiotec.com), SH2, SH3, and SH4 (culture supernatants obtained from hybridoma cells; American Type Culture Collection; Manassas, VA; http://www.atcc.org). The nonspecific mouse IgG (Vector; Burlingame, CA; http://www.vectorlabs.com) was substituted for the primary antibodies as an isotype control. The secondary antibody, anti-mouse IgG-fluorescein isothiocyanate (FITC) or IgG-phycoerythrin (PE) (Vector), was incubated with cells for 20 minutes at 4°C in the dark. The cells were then washed with PBS and resuspended in 1 ml of PBS for analysis. Data were analyzed using the fluorescence-activated cell sorter (FACS) FACSscan flow cytometer (Becton, Dickinson and Company; San Jose, CA; http://www.bd.com) with a minimum of 30,000 events.
Osteogenic and Adipogenic Differentiation
MSCs established from bone marrow and cord blood were seeded at about 7 x 103 cells/cm2 into six-well plates and grown to confluence. To induce osteogenic differentiation, cells were incubated in -MEM supplemented with 10% FBS, 0.1 µM dexamethasone (Sigma), 10 mM ß-glycerolphosphate (Sigma), and 50 µM ascorbic acid (Sigma) for 3 weeks. To initiate adipogenic differentiation, cells were incubated in -MEM supplemented with 10% FBS, 1 µM dexamethasone (Sigma), 0.5 mM methyl-isobutylxanthine (Sigma), 10 µg/ml insulin (Gibco-BRL; Carlsbad, CA; http://www.lifetech.com), and 100 µM indomethacin (Sigma) for 3–5 weeks. To assay the effect of leptin on osteogenic and adipogenic differentiation, human recombinant leptin (PeproTech; London, U.K.; http://www.peprotech.com) was added, at concentrations of 0.6 and 1.5 µg/ml, to each differentiation medium. After 3 weeks of culture, cells were analyzed for osteogenic and adipogenic features. Three replicates were tested for each sample.
Quantification and Detection of Osteogenic and Adipogenic Differentiation Potential
Cells were analyzed for alkaline phosphatase (ALPase) activity and Alizarin Red S staining (Sigma) to assess their osteogenic differentiation. ALPase activity was assayed following the manufacturer’s instruction (Sigma). Briefly, 1 ml of 0.05 N NaOH in ethanol was added to each well after ALPase activity stain [12]. The extraction was measured spectrophotometrically at 550 nm. The amount of ALPase activity was represented as a ratio compared with that of the undifferentiated cells. To quantify calcium deposition, cells were washed with PBS and incubated with 2% Alizarin Red S solution (preadjusted to a pH of 5.5 with 0.5% NH4OH) for 5 minutes [12]. After PBS washing, 1 ml phosphate buffer (8 mM Na2HPO4 + 1.5 mM KH2PO4) containing 10% cetylpyoidimium chloride (Sigma) was added and incubated overnight. The extracted solution of cell-bound Alizarin Red S was measured spectrophotometrically at 550 nm and compared with a standard titration curve [12].
Adipogenic differentiation was assessed by Oil Red O (Sigma) staining for 15 minutes at room temperature. Afterward, cells were washed completely with 60% isopropanol (Sigma), and 1 ml ethanol was added to each well to extract the cell-bound Oil Red O. The amount of Oil Red O released was determined spectrophotometrically at 550 nm with a reference of 650 nm and compared with an Oil Red O standard titration curve [12]. The extraction of cell-bound Alizarin Red S and Oil Red O was normalized to the average cell number at the time of dye extraction. A panel of wells was trypsinized in parallel to count the number of cells.
Measurement of mRNA Level by Real-Time Polymerase Chain Reaction
Total RNA from each sample was isolated using Trizol reagent (MRC; Cincinnati, OH, http://www.mrcgene.com/tri.htm) following the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1 µg total RNA using ImPro-II reverse transcriptase (Promega; Madison, WI; http://www.promega.com) with oligo-dT primer in 20 µl reaction mixture. The relative expression level of ß-actin was used as an internal control to normalize specific gene expression in each sample. Real-time polymerase chain reaction (PCR) using the ABI Prism 7000 Sequence Detection System (Applied Biosystems; Foster City, CA; http://www.appliedbiosystems.com) was performed with 2 µl of the single-stranded cDNA sample with SYBR Green PCR master mix (Applied Biosystems). The relative quantitation of marker genes (Table 1
) was performed according to the 
Ct method, as described elsewhere [13].
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RESULTS |
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). FACS analysis was used to characterize cell surface markers on bmMSCs and cbMSCs at passage 4. As shown in Figure 2, two typical profiles from bmMSCs and cbMSCs exhibited similar immunophenotypes, which were negative for CD34 (a hematopoietic stem cell marker) and CD45 (leukocyte common antigen) and positive for the mesenchymal progenitor–specific markers SH2, SH3, and SH4. These data are consistent with previous reports [3, 14].
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). After 3 weeks in culture, cbMSCs fully differentiated into osteoblasts. Comparatively, bmMSCs showed a relatively lower osteogenic tendency; only about 20% of the bmMSCs produced mineral nodules after 1 week of osteogenic induction and about 60% osteoblasts were found after 3 weeks of culture. The difference in the osteogenic differentiation capacity between cbMSCs and bmMSCs was further examined by Alizarin Red S staining and ALPase activity analysis (Fig. 3A). The amounts of Alizarin Red S and ALPase activity were 6.9-fold and 2.7-fold greater for cbMSCs than for bmMSCs in osteogenic culture, respectively (data not shown).
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). The adipogenic induction protocol used in this study demonstrated that bmMSCs had a markedly greater adipogenic tendency than cbMSCs. The amount of cell-bound Oil Red O in bmMSC adipogenic cultures was 17.3-fold higher than that found in cbMSC adipogenic cultures (data not shown).
Effects of Leptin on Osteogenesis and Adipogenesis
Leptin has been demonstrated to be an important factor in regulating bone and fat formation in humans [10, 11]. To evaluate whether leptin could affect the differentiation of MSCs in vitro, recombinant human leptin was added in combination with the differentiation media. After 3 weeks, osteogenesis was significantly enhanced by leptin in both bmMSCs and cbMSCs. Mineralization was estimated by the amount of cell-bound Alizarin Red S (Fig. 4A). The effect of leptin in increasing osteogenesis was also confirmed by ALPase activity staining (Fig. 4B). Interestingly, a dose-dependent enhancement of osteogenesis by leptin was only found in bmMSCs. bmMSCs treated with 0.6 µg/ml and 1.5 µg/ml of leptin had mineral accumulation levels that were 73% and 144% greater, respectively. The ALPase activity staining ratios were also 36% and 51% greater, respectively. However, treatment of cbMSCs with 0.6 µg/ml of leptin resulted in only a 25% greater mineral accumulation and a 13% greater ALPase activity staining ratio. Increasing the leptin concentration to 1.5 µg/ml had no effect; the leptin effect was seemingly saturated at the lower level, according to the analysis of mineral accumulation and ALPase activity staining in cbMSCs. These results indicate that leptin had a relatively stronger osteogenesis promoting effect in bmMSCs than in cbMSCs, although the osteogenic capacity of bmMSCs was still much lower than that of cbMSCs under leptin treatment.
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. Leptin significantly reduced adipogenesis in both bmMSCs and cbMSCs, as assessed by Oil Red O staining. When leptin was incorporated into the adipogenic media, both types of MSC yielded a lower number of lipid droplets. The accumulation of cell-bound Oil Red O was 61% and 68% lower in bmMSCs under treatment with 0.6 µg/ml and 1.5 µg/ml of leptin, respectively, whereas the cell-bound Oil Red O was 41% and 44% lower in cbMSCs under the same conditions. These results indicate that leptin had a relatively stronger suppressive effect on adipogenesis in bmMSCs than in cbMSCs, although the accumulated oil was still much higher in bmMSCs than cbMSCs under leptin treatment.
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2) Transcription2 are the major transcription factors for early osteogenesis and adipogenesis respectively.
Consistent with the Alizarin Red S staining and ALPase activity assay, leptin produced a dose-dependent increase in Cbfa1 expression during osteogenesis only in bmMSCs. Cbfa1 mRNA expression was 2.0-fold and 3.2-fold greater in bmMSCs under the 0.6 µg/ml and 1.5 µg/ml leptin treatments, respectively, at the first week of osteogenesis, while it was only 1.2-fold and 1.3-fold greater in cbMSCs under the same conditions (Fig. 4C).
During adipogenic induction, PPAR2 mRNA expression levels in bmMSCs treated with 0.6 µg/ml and 1.5 µg/ml leptin supplementation were only 48% and 41% of the levels observed without leptin treatment, respectively (Fig. 5B). In cbMSCs, the corresponding PPAR2 mRNA expression levels were 79% and 68% of the level seen without leptin supplementation, respectively. These results confirm that bmMSCs were significantly more sensitive to leptin than cbMSCs in osteogenic promotion and adipogenic suppression.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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At the same developmental stage, however, MSCs isolated from different tissues might exhibit diverse differentiation capacities. For example, MSCs isolated from different fetal tissues during the second trimester showed diverse adipogenic and osteogenic differentiation potentials [12]. The adipogenic capacity was markedly lower, but the osteogenic capacity was higher, in the MSCs derived from fetal spleen than from bone marrow, liver, or lung [12]. Bieback et al. [18] also reported a higher adipogenic potential in bmMSC clones than cbMSC clones. Thus, do MSCs from adult bone marrow and postnatal cord blood possess different differentiation tendencies? In this study, cbMSCs displayed an apparently higher tendency to undergo osteogenesis than bmMSCs did. During osteogenic differentiation, osteoblasts exhibited strong ALPase activity, followed by matrix maturation and then transition into the matrix mineralization period [19]. cbMSCs accumulated much more mineral nodules and expressed higher ALPase activity than bmMSCs under the same osteogenic induction conditions. In contrast, bmMSCs had more lipid droplet–containing cells than cbMSCs during adipogenesis. These findings indicate that cbMSCs have a stronger osteogenic potential but much lower adipogenic capacity than bmMSCs.
Although leptin has emerged as a potential contributor to bone turnover and fat mass, its effects on bone metabolism remain unclear. Some in vivo evidence has revealed that reducing the serum leptin level or the expression level of functional leptin receptor increases bone mass [20, 21]. Conversely, leptin has also been reported to have many positive effects on osteoblasts, by increasing cell proliferation, differentiation, and bone mineralization [10, 22]. Leptin also acts on muscle cells and chondrocytes in vitro [8, 9]. Not surprisingly, these cells are all derived from mesenchymal stem cells. Thomas et al. [23] reported that leptin could enhance osteogenesis and inhibit adipogenesis in hMS-12 cells, a conditionally immortalized human marrow stromal cell line. Our data demonstrate, for the first time, that leptin can act directly on primary MSCs to regulate mesenchyme-lineage differentiation in vitro, including promoting osteogenesis and suppressing adipogenesis. Moreover, bmMSCs are more sensitive to leptin than cbMSCs in both osteogenesis and adipogenesis. Because of the high leptin expression levels in human bone marrow adipocytes [24], leptin may serve as an endocrine modulator of MSC differentiation. These in vitro studies may be relevant to the clinical observation that obesity is associated with greater bone mineral density [25].
Our real-time PCR data are in agreement with the observation that osteogenesis and adipogenesis are activated by the transcriptional regulators Cbfa1 and PPAR2, respectively [26]. Leptin consistently showed a dose-dependent upregulation of Cbfa1 expression and downregulation of PPAR2 expression in bmMSCs during osteogenesis and adipogenesis, while leptin had only a slight effect on cbMSCs.
In conclusion, our data indicate that bmMSCs and cbMSCs possess heterogeneous differentiation potentials and different responses to leptin in vitro. These disparate characteristics of tissue-derived MSCs from different sources should be considered to better apply MSCs in vivo.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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, bmMSCs; 
, cbMSCs. * indicates statistically significant difference (p < 0.05) versus bmMSCs under the same conditions.